Anais da Academia Brasileira de Ciências (2011) 83(3): 1045-1058 (Annals of the Brazilian Academy of Sciences) Printed version ISSN 0001-3765 / Online version ISSN 1678-2690 www.scielo.br/aabc Study of the antiproliferative potential of seed extracts from Northeastern Brazilian plants PAULO MICHEL P. FERREIRA 1 , DAVI F. FARIAS 2 , MARTÔNIO P. VIANA 2 , TEREZINHA M. SOUZA 2 , ILKA M. VASCONCELOS 3 , BRUNO M. SOARES 4 , CLÁUDIA PESSOA 4 , LETÍCIA V. COSTA-LOTUFO 4 , MANOEL O. MORAES 4 and ANA F.U. CARVALHO 2 1 Departamento de Ciências Biológicas, Campus Senador Helvídio Nunes de Barros, Universidade Federal do Piauí, Rua Cícero Duarte, 905, 64600-000 Picos, PI, Brasil 2 Departamento de Biologia, Universidade Federal do Ceará, Av. Mister Hall, s/n, 60455-970 Fortaleza, CE, Brasil 3 Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Av. Mister Hall s/n, 60455-970 Fortaleza, CE, Brasil 4 Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, Rua Cel. Nunes de Melo, 1127, 60430-270, Fortaleza, CE, Brasil Manuscript received on August 14, 2009; accepted for publication on March 4, 2011 ABSTRACT This study assessed the antiproliferative and cytotoxic potential against tumor lines of ethanolic seed extracts of 21 plant species belonging to different families from Northeastern Brazil. In addition, some underlying mechanisms involved in this cytotoxicity were also investigated. Among the 21 extracts tested, the MTT assay after 72 h of incubation demonstrated that only the ethanolic extract obtained from Myracrodruon urundeuva seeds (EEMUS), which has steroids, alkaloids and phenols, showed in vitro cytotoxic activity against human cancer cells, being 2-fold more active on leukemia HL-60 line [IC 50 value of 12.5 (9.5-16.7) μg/mL] than on glioblastoma SF-295 [IC 50 of 25.1 (17.3-36.3) μg/mL] and Sarcoma 180 cells [IC 50 of 38.1 (33.5-43.4) μg/mL]. After 72h exposure, flow cytometric and morphological analyses of HL-60-treated cells showed that EEMUS caused decrease in cell number, volume and viability as well as internucleosomal DNA fragmentation in a dose-dependent way, suggesting that the EEMUS triggers apoptotic pathways of cell death. Key words: antiproliferative potential, Northeastern Brazilian plants, Myracrodruon urundeuva, sarcoma 180 tumor, seed extracts. INTRODUCTION There is considerable scientific and commercial inter- est in discovering new anticancer agents from natural product sources (Kinghorn et al. 2003). The potential of using natural products as anticancer agents was recog- nized in the 1950s by the U.S. National Cancer Institute (NCI) and since then several studies have given valuable contributions to the discovery of new naturally occur- ring anticancer agents. In the 1980s, the development of new screening technologies led the research for new anticancer agents in plants and other organisms, focus- Correspondence to: Dr. Paulo Michel Pinheiro Ferreira E-mail: [email protected] / [email protected]ing on the tropical and sub-tropical regions of the world (Cragg and Newman 2005). Brazil possesses the largest diversity of plant spe- cies in the world, but less than 10% have been evalu- ated with respect to their biological characteristics, and fewer than 5% have been subjected to detailed phyto- chemical studies (Luna et al. 2005). In Northeastern Brazil, a region with approximately 1,539,000 km 2 , with warm and dry climate, grows the peculiar xerophitic “Caatinga” vegetation (dry land vegetation). In Caatinga flora, there are almost 1000 vascular plant species and due to the extreme climate conditions most species are endemic (Lemos and Rodal 2002, Sampaio 2002, An Acad Bras Cienc (2011) 83 (3)
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Anais da Academia Brasileira de Ciências (2011) 83(3): 1045-1058(Annals of the Brazilian Academy of Sciences)Printed version ISSN 0001-3765 / Online version ISSN 1678-2690www.scielo.br/aabc
Study of the antiproliferative potential of seed extractsfrom Northeastern Brazilian plants
PAULO MICHEL P. FERREIRA1, DAVI F. FARIAS2, MARTÔNIO P. VIANA2,TEREZINHA M. SOUZA2, ILKA M. VASCONCELOS3, BRUNO M. SOARES4, CLÁUDIA PESSOA4,
LETÍCIA V. COSTA-LOTUFO4, MANOEL O. MORAES4 and ANA F.U. CARVALHO2
1Departamento de Ciências Biológicas, Campus Senador Helvídio Nunes de Barros, Universidade Federal do Piauí,Rua Cícero Duarte, 905, 64600-000 Picos, PI, Brasil
2Departamento de Biologia, Universidade Federal do Ceará, Av. Mister Hall, s/n, 60455-970 Fortaleza, CE, Brasil3Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará,
Av. Mister Hall s/n, 60455-970 Fortaleza, CE, Brasil4Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará,
Rua Cel. Nunes de Melo, 1127, 60430-270, Fortaleza, CE, Brasil
Manuscript received on August 14, 2009; accepted for publication on March 4, 2011
ABSTRACT
This study assessed the antiproliferative and cytotoxic potential against tumor lines of ethanolic seed extracts of 21plant species belonging to different families from Northeastern Brazil. In addition, some underlying mechanismsinvolved in this cytotoxicity were also investigated. Among the 21 extracts tested, the MTT assay after 72 h ofincubation demonstrated that only the ethanolic extract obtained from Myracrodruon urundeuva seeds (EEMUS),which has steroids, alkaloids and phenols, showed in vitro cytotoxic activity against human cancer cells, being 2-foldmore active on leukemia HL-60 line [IC50 value of 12.5 (9.5-16.7) μg/mL] than on glioblastoma SF-295 [IC50 of 25.1(17.3-36.3) μg/mL] and Sarcoma 180 cells [IC50 of 38.1 (33.5-43.4) μg/mL]. After 72h exposure, flow cytometricand morphological analyses of HL-60-treated cells showed that EEMUS caused decrease in cell number, volume andviability as well as internucleosomal DNA fragmentation in a dose-dependent way, suggesting that the EEMUS triggersapoptotic pathways of cell death.
There is considerable scientific and commercial inter-est in discovering new anticancer agents from naturalproduct sources (Kinghorn et al. 2003). The potential ofusing natural products as anticancer agents was recog-nized in the 1950s by the U.S. National Cancer Institute(NCI) and since then several studies have given valuablecontributions to the discovery of new naturally occur-ring anticancer agents. In the 1980s, the developmentof new screening technologies led the research for newanticancer agents in plants and other organisms, focus-
ing on the tropical and sub-tropical regions of the world(Cragg and Newman 2005).
Brazil possesses the largest diversity of plant spe-cies in the world, but less than 10% have been evalu-ated with respect to their biological characteristics, andfewer than 5% have been subjected to detailed phyto-chemical studies (Luna et al. 2005). In NortheasternBrazil, a region with approximately 1,539,000 km2, withwarm and dry climate, grows the peculiar xerophitic“Caatinga” vegetation (dry land vegetation). In Caatingaflora, there are almost 1000 vascular plant species anddue to the extreme climate conditions most species areendemic (Lemos and Rodal 2002, Sampaio 2002,
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Santos et al. 2008). Despite this great biodiversity,Northeastern Brazilian plants are relatively under-exploited with regard to discoveries of active biologicalsubstances (Luna et al. 2005).
Seeds, especially from legumes, are recognized bytheir nutritional value, rich in proteins, carbohydratesand oil, though they are not merely a site to accumu-late organic materials. They need physical and chem-ical mechanisms for protection and/or defense for thedeveloping embryo. The compounds involved in chem-ical defense include lectins, protease and amylase in-hibitors, toxins and low molecular mass compounds (sec-ondary metabolites) (Xavier-Filho 1993, Sampaio et al.1992, Ferreira et al. 2009). According to some reportsmany seeds and other parts from Northeastern Brazil-ian plants are exploited in popular medicine and manyof these present important pharmacological properties.The ethnomedical data and some pharmacological activ-ities of the studied plants are shown in Table I. Never-theless, these plants, especially their seeds, are scarcelystudied concerning cytotoxicity on tumor cell strains.Thus, the aim of the present study was to assess theantiproliferative potential of ethanolic seed extracts oftwenty-one plant species belonging to different familiesfrom Northeastern Brazil on tumor cells and study someunderlying mechanisms involved in this cytotoxicity.
MATERIALS AND METHODS
ANIMALS
Adult Swiss mice (Mus musculus Linnaeus, 1758) wereobtained from the animal facilities of the UniversidadeFederal do Ceará, Fortaleza, Brazil. They were kept inwell ventilated cages under standard conditions of light(12 h with alternative day and night cycles) and tem-perature (27 ± 2◦C) and housed with access to com-mercial rodent stock diet (Nutrilabor, Campinas, Brazil).All procedures are in accordance with COBEA (Colé-gio Brasileiro de Experimentação Animal) (Process no.102/2007) and International Standard on the care anduse of experimental animals (EEC Directive of 1986,86/609/EEC).
PLANT MATERIAL
Harvest of the plant materials was carried out in threedifferent localities in Ceará State, Northeastern Brazil:
the Caatinga forest (dry land vegetation), Araripe Na-tional Forest (rain forest), and in the coastal zone. Ma-ture wild seeds (at least 500 g) of each plant specieswere collected during the dry period (from January 2005to November 2007), with help of native people. Plantswere identified and voucher specimens were depositedat Herbarium Prisco Bezerra – EAC, Universidade Fed-eral do Ceará (Fortaleza, Ceará, Brazil). Table I de-scribed all the species studied in this work, their vouchernumbers, common names, harvest data and medicinalapplications.
PREPARATION OF CRUDE EXTRACTS
Seeds of freshly collected plant material were separated,immediately air dried and finally ground in a laborat-ory mill (Quimis, Campinas, São Paulo, Brazil) to amoderately-fine powder (mesh size 0.5 mm). Powderedmaterial (500 g) was submitted to extraction with 99%ethanol (1.5 L) at room temperature (25-27◦C) for 3days and filtered. The residue was re-extracted twicein a similar manner. The extracts were evaporated andbulked under reduced pressure in a rotary evaporator.Crude extracts were stored in a freezer at –20◦C un-til required. A stock solution containing 10 mg/mL ofeach crude extract was prepared by suspending 10 mg ofextract in 1 mL of sterile dimethylsulphoxide (DMSO,Sigma Aldrich) (Torres et al. 2005, Costa et al. 2008,Buriol et al. 2009, Magalhães et al. 2010), mixed bysonication (Bandelin, model RK-100, Berlin, GER)for 20 min.
CYTOTOXICITY AGAINST HUMAN TUMOR CELL LINES
The antiproliferative potential of the seed extracts wasevaluated by the MTT assay (Mosmann 1983) against4 human tumor cell lines: HL-60 (leukemia), SF-295(glioblastoma), HCT-8 (colon) and MDA/MB-435(melanoma), all obtained from the National Cancer In-stitute (Bethesda, MD, USA). All cell lines were main-tained in RPMI 1640 medium supplemented with 10%fetal bovine serum, 2 mM glutamine, 100 U/mL pen-incillin and 100 μg/mL streptomycin, at 37◦C with 5%CO2. Tumor cell growth was quantified by the ability ofliving cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H -tetrazolium bromide (MTT)to a purple formazan product. Briefly, cells were platedin 96-well plates [0.7 × 105 cells/well for adherent cells
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TABLE IPlant species employed in this study and some ethnobotanical uses and pharmacological activities.
FAMILYBotanical nameCommon name Yield (%) Traditional medicinal use Pharmacological activities[Locality: date of collect]Voucher number
ANACARDIACEAEMyracrodruon urundeuva Fr. All.Aroeira-do-sertão[Araripe National Forest: 01/07]34,865
25.2 Cutaneous and gynecologicalaffections, kidney and respiratoryproblems; anti-inflammatory,antiulcer, healing (Sousa etal. 2004); antimicrobial (Matos2000); analgesic, anti-diarrheal(Maia 2004)
Anti-colitis (Rodrigues et al.2002); anti-inflammatory,analgesic (Viana et al. 2003);anti-ulcer (Souza et al. 2007);anti-periodontitis (Botelho etal. 2007)
Schinopsis brasiliensis Engl.Braúna[Araripe National Forest: 01/07]35,643
2.88 Cause nervosism and hysteria,analgesic (Maia 2004)
Not described
CARYOCARACEAECaryocar coriaceum Wittm.Pequi[Araripe National Forest: 09/05]35.231
Dioclea megacarpa RolfeMucunã, olho-de-boi[Araripe National Forest: 01/05]38,110
2.40 Not described Not described
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TABLE I (continuation)
FAMILYBotanical nameCommon name Yield (%) Traditional medicinal use Pharmacological activities[Locality: date of collect]Voucher number
FABACEAEEnterolobium contortisiliquum (Vell.) MorongOrelha-de-negro[Araripe National Forest: 09/05]38,115
2.80 Not described Antimicrobial (Shahat et al. 2008);cytotoxic (Mimaki et al. 2003);anticoagulant (Sampaio et al.1992); pro-inflammatory (Castro-Faria-Neto et al. 1991); hemolytic(De Sousa and Morhy 1989)
Hymenaea courbaril L.Jatobá[Araripe National Forest: 01/05]38,108
9.50 Not described 5-lipoxygenase inhibition (Bragaet al. 2000); antiplasmodial(Köhler et al. 2002)
10.7 Not described Antifungal (Tsuzuki et al. 2007);antiulcer (Albiero et al. 2008)
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(SF295, MDA/MB-435, HCT-8) and 0.3×105 cells/wellfor suspended cells (HL-60)] and extracts (50 μg/mL)were added to each well. After 72 h of incubation,the supernatant was replaced by fresh medium contain-ing MTT (0.5 mg/mL), the formazan product was dis-solved in 150 μL DMSO and absorbance was measuredat 595 nm (DTX 880 Multimode Detector, BeckmanCoulter). Doxorubicin (0.3 μg/mL, Sigma Aldrich) wasused as positive control.
HEMATOXYLIN-EOSIN STAIN
HL-60 cells treated or untreated with the ethanolicextract of Myracrodruon urundeuva seeds (EEMUS)(6.25, 12.5 and 25 μg/mL) were examined for morpho-logical changes by light microscopy (Metrimpex Hun-gary/PZO-Labimex Modelo Studar lab) after 72 h ex-posure. Cells were harvested, transferred to cytospinslides, fixed with methanol for 1 min and stained withHematoxylin-Eosin (H&E). Doxorubicin (0.3 μg/mL)was used as positive control.
FLOW CYTOMETRY ANALYSES
All cytometry analyses were determined in a GuavaEasyCyte Mine (Guava Express Plus software). Fivethousand events were evaluated per experiment and cel-lular debris was omitted from the analysis. Experimentswere performed in triplicate using HL-60 cells and ana-lyzed after 72 h of incubation with EEMUS (6.25, 12.5and 25 μg/mL).
Cell number and membrane integrity
Cell membrane integrity was evaluated by the exclusionof propidium iodide (50 μg/mL, Sigma Aldrich Co. –St. Louis, MO/USA). Briefly, 100 μL of treated anduntreated cells were incubated with propidium iodide(50 μg/mL). The cells were then incubated for 5 min.Fluorescence was measured and cell number andmembrane integrity were determined (Darzynkiewiczet al. 1992).
Internucleosomal DNA fragmentation
Internucleosomal DNA fragmentation was evaluatedby the incorporation of propidium iodide (50 μg/mL).Briefly, HL-60 cells were treated and then incubated at
25◦C for 30 min, in the dark, in a lysis solution con-taining 0.1% citrate, 0.1% Triton X-100 and 50 μg/mLpropidium iodide. Fluorescence was measured andDNA fragmentation was analyzed according to Nicolettiet al. (1991).
Measurement of mitochondrial transmembranepotential
Mitochondrial transmembrane potential was determinedby the retention of rhodamine 123 dye. Aliquots re-moved from wells were incubated with 200 μL ofrhodamine 123 in the dark for 15 min and centrifugedat 2000 rpm/5 min. Subsequently, cells were harvestedand incubated in PBS solution for 30 min. at 25◦C (Cury-Boaventura et al. 2003).
In vitro ANTIPROLIFERATIVE ACTIVITY ON
SARCOMA 180 CELLS
In order to predict activity of EEMUS towards an invivo cancer model, ascite-bearing female mice between7 and 9 days postinoculation were sacrificed by cervicaldislocation and a suspension of Sarcoma 180 cells washarvested from the intraperitoneal cavity under asepticconditions. The suspension was centrifuged at 500 X gfor 5 min to obtain a cell pellet and washed three timeswith RPMI medium. Cell concentration was adjustedto 0.5 × 106 cells/mL in RPMI 1640 medium supple-mented with 20% fetal bovine serum, 2 mM glutamine,100 U/mL penincillin and 100 μg/mL streptomycin,plated in a 96-well plate and incubated with increasingconcentrations of EEMUS (1.56-100 μg/mL). Cell pro-liferation was determined by the Alamar Blue assay after72 h extract exposure according to Ferreira et al. (2010),with some modifications. Eight hours before the end ofthe incubation, 10 μL of stock solution (0.312 mg/mL) ofAlamar Blue (Resazurin, Sigma Aldrich Co) were addedto each well. The absorbance was measured at 570 nmand 595 nm using a multiplate reader (DTX 880 Multi-mode Detector) and the drug effect was quantified as thepercentage of the control. Doxorubicin (0.3 μg/mL) wasused as positive control.
PHYTOCHEMICAL STUDY OF ETHANOLIC EXTRACTS
Phytochemical tests to detect the presence of secondarymetabolites in EEMUS such as phenols, tannis, leuco-anthocianidins, flavonoids, steroids, triterpens and alka-
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loids were performed according to Matos (2000). Thesetests are based on visual observation of color modifica-tion or precipitate formation after addition of specificreagents.
STATISTICAL ANALYSIS
For cytotoxicity assays, the IC50 and EC50 values andtheir 95% confidence intervals were obtained by non-linear regression using the Graphpad program (IntuitiveSoftware for Science, San Diego, CA). In order to de-termine differences, data (mean ± standard error mean)were compared by analysis of variance (ANOVA) fol-lowed by Newman-Keuls test (P<0.05).
RESULTS AND DISCUSSION
Drug discovery from medicinal plants has played animportant role in the treatment of cancer and most newclinical applications of plant secondary metabolites andtheir derivatives have applied towards combating cancer(Butler 2004, Cragg and Newman 2005). In this work,of the 21 ethanolic extracts tested, the analyses by MTTassay showed that only the ethanolic extract obtainedfrom M. urundeuva seeds showed cytotoxic potentialagainst cancer cells (Table II), given that it was the soleextract that caused cell proliferation inhibition higherthan 90% (Torres et al. 2005). Subsequently, we deter-mined its IC50 values on tumor lines by MTT assay ina similar way as described above with the bioactive ex-tract concentration ranging from 0.09 to 50 μg/mL. Ac-cording to the American National Cancer Institute, theIC50 limit to consider a promising crude extract forfurther purification is a value lower than 30 μg/mL (Suff-ness and Pezzuto 1990). As seen in Table III, EEMUSwas inactive against in vitro colon and melanoma tu-mors, while it demonstrated moderate activity on glio-blastoma [SF-295, IC50 of 25.1 (17.3.3-36.3) μg/mL]and especially on leukemia [HL-60, IC50 of 12.5 (9.5-16.7) μg/mL] cells. On the other hand, the positive con-trol doxorubicin presented high cytotoxicity against allcell lines (Table III).
Myracrodruon urundeuva Fr. Allemao, 1881(Anarcadiaceae), an endemic tree in NortheasternCaatinga, is a folk medicinal plant known as “aroeirado sertão” very used for treating bleeding gums andgynecological disorders (Viana et al. 2003, Monteiro etal. 2006). Different parts of M. urundeuva also possess
hepatoprotective, anti-diarrheal, anti-ulcer, cicatrizing,colonic anastomotic wound healing properties and lar-vicidal activity (Viana et al. 2003, Goes et al. 2005,Souza et al. 2007, Sá et al. 2009). Recently, Sá et al.(2008) isolated a termiticidal lectin from M. urundeuvaheartwood resistant to enzyme degradation to elucidatethe resistance and durability of its wood to biodegrada-tion by termites, a plague that has caused damages inseveral wooden parts in buildings and serious econom-ical issues. Despite its diverse pharmacological applica-tions, this is the first study showing a directed anticancerpotentiality of this plant.
In an attempt to envisage an antitumor action uponin vivo assessments, it was determined the EEMUS ac-tivity on Sarcoma 180 cells using the Alamar Blue assay.Here, we also found antiproliferative action of EEMUSagainst these malignant cells, exhibiting an IC50 of 38.1(33.5-43.4) μg/mL, an additional findings that stimu-lates further investigation to understand its mechanismof action.
Cell type antiproliferative specificity is observedin some plant extracts and this is probably due to thepresence of different classes of compounds (Cragg et al.1994). Hence, the use of more than one cell line is con-sidered necessary for detection of cytotoxic compounds.In the present work, EEMUS showed activity againstleukemia line 2-fold higher than against melanoma andSarcoma 180 cells (P<0.05). Then, we chose the HL-60 line to study underlying mechanisms involved in thein vitro cytotoxicity. The human HL-60 cell line, acutepromyelocytic leukemia with prevailing of neuthophilicpromyelocytes, is commonly used in the research fornovel cytotoxic substances (Costa et al. 2008, Ferreiraet al. 2010, Magalhães et al. 2010).
Extract-induced morphological alterations in HL-60 treated and untreated cells were examined following72 h of treatment and staining by H&E. In compari-son with the control cells and treated cells at the lowestconcentration (6.25 μg/mL), both exhibiting a typicalnon-adherent morphology and dividing cells (Fig. 1A),EEMUS-treated cells at 12.5 μg/mL presented chroma-tin condensation and shrinking (Fig. 1D) while damageon plasmatic membrane was mainly seen at 25 μg/mL(Fig. 1E). Doxorubicin also induced reduction in cellvolume, besides nuclear disintegration and chromatincondensation (Fig. 1B).
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TABLE IITumor cell proliferation inhibition (%) of ethanolic seed extracts of twenty-one plant species belonging
to different families from Northeastern Brazil determined by MTT assay after 72h of incubationat the concentration of 50 μg/mL.
*Results are expressed as mean ± standard error mean (S.E.M.) from two independent experiments for leukemia (HL-60),melanona (MDA/MB-435), glioblastoma (SF-295) and colon (HCT-8) human cancer cells. All cell lines were plated with RPMI1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penincillin and 100 μg/mL streptomycin,at 37◦C with 5% CO2. Doxorubicin (0.3 μg/mL) was used as positive control.
TABLE IIIIn vitro cytotoxic activity of the ethanolic extract of Myracrodruon
urundeuva seeds (EEMUS) against human tumor cell linesdetermined by MTT assay after 72 h exposure.
SubstanceIC50 (μg/mL)∗
HL-60 MDA/MB-435 SF-295 HCT-8
Doxorubicin0.02 0.48 0.23 0.01
0.01–02 0.34–0.66 0.19–0.25 0.01–0.02
EEMUS12.5
>5025.1
>509.5–16.7 17.3–36.3
*Data are presented as IC50 values and 95% confidence intervals for leukemia (HL-60), melanoma (MDA/MB-435), glioblastoma (SF-295) and colon (HCT-8) cells.All cell lines were plated with RPMI 1640 medium supplemented with 10% fetalbovine serum, 2 mM glutamine, 100 U/mL penincillin and 100 μg/mL streptomycin,at 37◦C with 5% CO2. Doxorubicin (0.009-5 μg/mL) was used as positive control.Independent experiments were performed in triplicate.
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Fig. 1 – Morphological analysis of 72 h-untreated (A) or treated leukemia HL-60 cells with ethanolic extract of Myracrondruon urundeuva
seeds (EEMUS) 6.25 (C), 12.5 (D) and 25 μg/mL (E), stained by Hematoxylin-Eosin (H&E) and examined by light microscopy. Doxorubicin
(0.3 μg/mL) was used as positive control (B). Negative control (A) was treated with the vehicle used for diluting the tested substance. Black arrows:
chromatin condensation; white arrows: cell volume reduction; black dashed arrow: plasmatic membrane disruption. Magnification, 400×.
Growth-inhibitory effects of the EEMUS were alsoanalyzed in HL-60-treated cells by flow cytometry. Thepropidium iodide intercalation test showed that EEMUScaused a decreasing in the cell number in a dose-de-pendent manner after 72 h of exposure (Fig. 2A), start-ing at the concentration of 12.5 μg/mL (IC50 value).These results are consistent with the MTT findings andwith reduction of cell viability (Fig. 2B) at 25 μg/mL(71.9 ± 2.4%) in comparison to the negative control(95.1 ± 1.1%) (P<0.01). Internucleosomal DNA evalu-ations showed that EEMUS led to a significant and dose-dependent increase in the DNA fragmentation (P<0.01),with fragmentation percentages of 27.7 ± 2.4% and45.5 ± 2.0%, for the concentrations of 12.5 and 25 μg/
mL, respectively (Fig. 3). On the other hand, statisticallysignificant cellular mitochondrial depolarization (Fig.2B) was detected only at the highest concentration 27.6± 4.5% (P<0.01), suggesting that cell death should beinitially triggered by a mitochondrial independent path-way or it could be an apoptosis resulting from an earlyDNA fragmentation (Wang et al. 1999).
Morphological and flow cytometric studies re-vealed a concomitant deoxyribonucleic acid disintegra-tion (increasing sub-G1 population cells), chromatin con-densation with membrane integrity at 12.5 μg/mL andmembrane disruption at higher concentrations. Theseresults suggest dose-dependent apoptotic cell death ac-tivation by EEMUS, though other biochemical and
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Fig. 2 – Effects of the ethanolic extract of Myracrodruon urundeuva seeds (EEMUS) on HL-60 cells analyzed by flow cytometry after 72 h
exposure. A – Total of cells; B – Cell membrane integrity. Analyses were determined by exclusion of propidium iodide. Negative control (C)
was treated with the vehicle used to dilute the tested substance. Doxorubicin (0.3 μg/mL) was used as positive control (D). Results are expressed
as mean ± standard error of measurement (S.E.M.) from three independent experiments. *P<0.01 compared to control by ANOVA followed by
Student Newman-Keuls test.
morphological examinations are necessary to confirm it,such as phosphatidylserine externalization and caspasedeterminations (Strasser et al. 2000).
The phytochemical analyses detected the presence
of steroids, alkaloids and phenols in EEMUS (data notshown). Some steroids, flavonoids and other phenolicsubstances are frequently associated with the aging pro-cess of the human body, production of free radicals due
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Fig. 3 – DNA fragmentation on HL-60 cells determined by flow cytometry after 72 h of incubation with ethanolic extract of Myracrodruon
urundeuva seeds (EEMUS). All evaluations were performed by nuclear fluorescence using propidium iodide, triton X-100 and citrate. Negative
control (C) was treated with the vehicle used for diluting the tested substance. Doxorubicin (0.3 μg/mL) was used as positive control (D). Results
are expressed as mean ± standard error of measurement (S.E.M.) from three independent experiments. *P<0.01 compared to control by ANOVA
followed by Student Newman-Keuls test.
to metabolic processes, initiation and promotion of can-cer and tissue injury by free radicals, which has inducedthe intake of antioxidant products as chemical factorsthat prevent the onset of diseases (Núñez-Sellés 2005).Previously, it was demonstrated that hydroalchoolicstem bark extracts from M. urundeuva exert anti-inflam-matory effects attributed to chalcones (Viana et al. 2003,Souza et al. 2007), a compound belonging to the groupof flavonoids naturally found in fruits, flowers, vegeta-bles, teas and wines (Abdulla and Gruber 2000, Ferreiraet al. 2008). Besides the traditional antioxidant prop-erties attributed to the flavonoids, some chalcones andtheir derivatives have reported to be potent cyclooxyge-nase inhibitors (Hsieh et al. 1998). This is an important
approach to some kinds of cancers, since COX-2 block-age avoids the expression of NF-κB activation, a keynuclear transcription factor involved in controlling in-flammation and tumorigenesis (Surh et al. 2001), sinceinflammation has been frequently found in premalignantlesions (Dranoff 2004).
This study displays the antiproliferative action ofthe ethanolic extract of Myracrondruon urundeuva seedson leukemia cells by death suggestive of apoptosis andalso showed its potential against experimental in vivo tu-mors. Further studies to support these discoveries are inprogress as well as phytochemical and molecular invest-igations to identify the bioactive compound(s) responsi-ble for this cytotoxic activity.
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ACKNOWLEDGMENTS
We wish to thank Conselho Nacional de Desenvol-vimento Científico e Tecnológico (CNPq), Coordena-ção de Aperfeiçoamento de Pessoal de Nível Superior(CAPES), Fundação Cearense de Apoio ao Desenvol-vimento Científico e Tecnológico (FUNCAP), Finan-ciadora de Estudos e Projetos (FINEP) and Banco doNordeste do Brasil (BNB) for financial support in theform of grants and fellowship awards. We are gratefulto Berenice Alves and Silvana França dos Santos fortechnical assistance.
RESUMO
Este estudo avaliou o potencial antiproliferativo e citotóxico
contra linhagens de células tumorais de extratos etanólicos
de sementes de vinte e uma espécies vegetais pertencentes a
diferentes famílias do Nordeste brasileiro. Além disso, al-
guns mecanismos subjacentes envolvidos nesta citotoxidade
também foram investigados. Dentre os 21 extratos testados
pelo ensaio do MTT após 72 h de incubação, apenas o ex-
trato etanólico obtido a partir de sementes de Myracrodruon
urundeuva (EEMUS), o qual apresentou traços de esteróides,
alcalóides e fenóis em sua composição, demonstrou atividade
citotóxica in vitro contra células tumorais humanas, sendo 2
vezes mais ativo sobre a linhagem leucêmica HL-60 [IC50 valor
de 12,5 (9,5-16,7) μg/mL] do que sobre células de glioblas-
toma SF-295 [IC50 de 25,1 (17,3-36,3) μg/mL] e de sarcoma
180 [IC50 de 38,1 (33,5-43,4) μg/mL]. Após 72 h de exposição,
as análises morfológicas e por citometria de fluxo de células
HL-60 tratadas com EEMUS mostraram diminuição no núme-
ro de células, seu volume e viabilidade, assim como fragmen-
tação internucleosomal do DNA de forma dose-dependente,
sugerindo que a ação antiproliferativa de EEMUS pode ser
ativada por vias apoptóticas.
Palavras-chave: potencial antiproliferativo, plantas do Nor-
deste Brasileiro, Myracrodruon urundeuva, tumor sarcoma
180, Extratos de sementes.
REFERENCES
ABDULLA M AND GRUBER P. 2000. Role of diet modifica-tion in cancer prevention. Biofactors 12: 45–51.
ALBIERO ALM, SERTIÉ JAA AND BACCHI EM. 2008. An-tiulcer activity of Sapindus saponaria L. in the rat. JEthnopharmacol 82: 41–44.
NA, CAVADA BS AND VALE MR. 2005. Anti-inflam-matory and antimicrobial effect of lectin from Loncho-carpus sericeus seeds in an experimental rat model ofinfectious peritonitis. J Pharm Pharmacol 57: 919–922.
MM AND BOLOGNESE AM. 2008. In vitro antioxidantpotential of medicinal plant extracts and their activitiesagainst oral bacteria based on Brazilian folk medicine.Arch Oral Biol 53: 545–552.
BOTELHO MA, RAO VS, CARVALHO CB, BEZERRA-FI-LHO JG, FONSECA SG, VALE ML, MONTENEGRO D,CUNHA F, RIBEIRO RA AND BRITO GA. 2007. Lippiasidoides and Myracrodruon urundeuva gel prevents alve-olar bone resorption in experimental periodontitis in rats.J Ethnopharmacol 113: 471–478.
BRAGA FC, WAGNER H, LOMBARDI JA AND DE OLIVEIRA
AB. 2000. Screening Brazilian plant species for in vitroinhibition of 5-lipoxygenase. Phytomed 6: 447–452.
BURIOL L ET AL. 2009. Composição química e atividadebiológica de extrato oleoso de própolis: uma alternativaao extrato etanólico. Quim nova 32: 296–302.
BUTLER MS. 2004. The role of natural product chemistry indrug discovery. J Nat Prod 67: 2141–2153.
CASTRO-FARIA-NETO HC ET AL. 1991. Pro-inflammatoryactivity of enterolobin: a haemolytic protein purified fromseeds of the Brazilian Tree Enterolobium contortisiliquum.Toxicon 29: 1143–1150.
COSTA PM, FERREIRA PMP, BOLZANI VS, FURLAN M,SANTOS VAFFM, CORSINO J, MORAES MO, COSTA-LOTUFO LV, MONTENEGRO RC AND PESSOA C. 2008.Antiproliferative activity of pristimerin isolated fromMaytenus ilicifolia (Celastraceae) in human HL-60 cells.Toxicol in vitro 22: 854–863.
COSTA-LOTUFO LV ET AL. 2003. Antiproliferative effectsof several compounds isolated from Amburana cearensisA. C. Smith. Z. Naturforsch. [C] 58: 675–680.
CRAGG GM, BOYD MR, CARDELLINA JH, NEWMAN DJ,SNADER KM AND MCCLOUD TG. 1994. Ethnobotanyand drug discovery experience of the US National CancerInstitute. In: CHADWICK DJ AND MARSH J (Eds), CibaFoundation Ethnobotany and the search for new drugs.Chichester: J Wiley & Sons, p. 178–196.
CRAGG GM AND NEWMAN DJ. 2005. Plants as a source ofanti-cancer agents. J Ethnopharmacol 100: 72–79.
CRUZ MC, SANTOS PO, BARBOSA AMJR, DE MELO
DL, ALVIANO CS, ANTONIOLLI AR, ALVIANO DSAND TRINDADE RC. 2007. Antifungal activity ofBrazilian medicinal plants involved in popular treatment
An Acad Bras Cienc (2011) 83 (3)
“main” — 2011/7/13 — 11:43 — page 1056 — #12
1056 PAULO MICHEL P. FERREIRA et al.
of mycoses. J Ethnopharmacol 111: 409–412.
CUNHA GM, FONTENELE JB, NOBRE-JÚNIOR HV, DE
SOUSA FC, SILVEIRA ER, NOGUEIRA NA, MORAES
MO, VIANA GS AND COSTA-LOTUFO LV. 2003. Cyto-toxic activity of chalcones isolated from Lonchocarpussericeus (Pocr.) Kunth. Phytother Res 17: 155–159.
CURY-BOAVENTURA MF, POMPÉIA C AND CURI R. 2003.Comparative toxicity of oleic acid and linoleic acid onJurkat cells. Clin Nutr 23: 721–732.
DARZYNKIEWICZ Z, BRUNO S, DEL BINO G, GORCZYCA
W, HOTZ MA, LASSOTA P AND TRAGANOS F. 1992.Features of apoptotic cells measured by flow cytometry.Cytometry 13: 795–808.
DE SOUSA MV AND MORHY L. 1989. Enterolobin, a hemo-lytic protein from Enterolobium contortisiliquum seeds(Leguminosae – Mimosoideae). Purification and charac-terization. An Acad Bras Cienc 61: 405–412.
DRANOFF G. 2004. Cytokines in cancer pathogenesis andcancer therapy. Nature Rev Cancer 4: 11–22.
EEC DIRECTIVE OF 1986. Council Directive of 24 Novem-ber 1986 on the approximation of laws, regulations andadministrative provisions of the Member States regardingthe protection of animals used for experimental and otherscientific purposes (86/609/EEC).
FERNANDES J, CASTILHO RO, DA COSTA MR, WAGNER-SOUZA K, COELHO-KAPLAN MA AND GATTASS CR.2003. Pentacyclic triterpenes from Chrysobalanaceaespecies: cytotoxicity on multidrug resistant and sensitiveleukemia cell lines. Cancer Lett 190: 165–169.
AMC AND MACHADO-NETO JG. 2009. Larvicidal act-ivity of the water extract of Moringa oleifera seedsagainst Aedes aegypti and its toxicity upon laboratoryanimals. An Acad Bras Cienc 81: 207–216.
FERREIRA PMP, SANTOS AG, TININIS AG, COSTA PM,CAVALHEIRO AJ, BOLZANI VS, MORAES MO, COS-TA-LOTUFO LV, MONTENEGRO RC AND PESSOA C.2010. Casearin X exhibits cytotoxic effects in leukemiacells triggered by apoptosis. Chem Biol Interac 188:497–504.
GOES AC, RODRIGUES LV, DE MENEZES DB, GRANGEIRO
MP AND CAVALCANTE AR. 2005. Histologic analysisof colonic anastomotic healing, in rats, under the actionof 10% Aroeira-do-sertao (Myracrodruon urundeuva Fr.All.) enema. Acta Cir Bras 20: 144–151.
HSIEH HK, LEE TH, WANG JP, WANG JJ AND LIN
CN. 1998. Synthesis and anti-inflammatory effect ofchalcones and related compounds. Pharm Res 15: 39–46.
KINGHORN AD ET AL. 2003. Novel strategies for the dis-covery of plant-derived anticancer agents. Pharm Biol 41:53–67.
KÖHLER I, JENETT-SIEMS K, SIEMS K, HERNÁNDEZ MA,IBARRA RA, BERENDSOHN WG, BIENZLE U AND
EICH E. 2002. In vitro antiplasmodial investigation ofmedicinal plants from El Salvador. Z Naturforsch [C] 57:277–281.
LEAL LK, COSTA MF, PITOMBEIRA M, BARROSO VM,SILVEIRA ER, CANUTO KM AND VIANA GS. 2006.Mechanisms underlying the relaxation induced by iso-kaempferide from Amburana cearensis in the guinea-pigisolated trachea. Life Sci 79: 98–104.
ER AND VIANA GS. 2008. Protective effects of am-buroside A, a phenol glucoside from Amburana cearensisagainst CCl4-induced hepatotoxicity in rats. Planta Med74: 497–502.
LEAL LK, NECHIO M, SILVEIRA ER, CANUTO KM, FON-TENELE JB, RIBEIRO RA AND VIANA GS. 2003. Anti-inflammatory and smooth muscle relaxant activities ofthe hydroalcoholic extract and chemical constituents fromAmburana cearensis A. C. Smith. Phytother Res 17: 335–340.
LEAL LK, NOBRE-JÚNIOR HV, CUNHA GM, MORAES
MO, PESSOA C, OLIVEIRA RA, SILVEIRA ER, CANU-TO KM AND VIANA GS. 2005. Amburoside A, a glu-coside from Amburana cearensis, protects mesencephaliccells against 6-hydroxydopamine-induced neurotoxicity.Neurosci Lett 388: 86–90.
LEMOS JR AND RODAL MJN. 2002. Fitossociologia docomponente lenhoso de um trecho da vegetação de caatin-ga no Parque Nacional Serra da Capivara, Piauí, Brasil.Acta Bot Bras 16: 23–42.
LUNA JS, SANTOS AF, LIMA MRF, OMENA MC, MEN-DONÇA FAC, BIEBER LW AND SANT’ANA AEG. 2005.A study of the larvicidal and molluscicidal activities ofsome medicinal plants from Northeast Brazil. J Ethno-pharmacol 97: 199–206.
MAGALHÃES HIF, FERREIRA PMP, MOURA ES, TORRES
MR, ALVES APNN, PESSOA ODL, COSTA-LOTUFO
LV, MORAES MO AND PESSOA C. 2010. In vitro and invivo antiproliferative activity of Calotropis procera stemextracts. An Acad Bras Cienc 82: 407–416.
MAIA GN. 2004. Caatinga: Árvores e arbustos e suas utili-dades. D & Z. Fortaleza, 413 p.
An Acad Bras Cienc (2011) 83 (3)
“main” — 2011/7/13 — 11:43 — page 1057 — #13
ANTIPROLIFERATIVE POTENTIAL OF NORTHEASTERN BRAZILIAN SEEDS 1057
MATOS FJA. 2000. Plantas Medicinais – guia de seleção eemprego de plantas usadas em fitoterapia no nordeste doBrasil. Imprensa Universitária. Fortaleza, 346 p.
MIMAKI Y, HARADA H, SAKUMA C, HARAGUCHI M, YUI
S, KUDO T, YAMAZAKI M AND SASHIDA Y. 2003. En-terolosaponins A and B, novel triterpene bisdesmosidesfrom Enterolobium contortisiliquum and evaluation fortheir macrophage-oriented cytotoxic activity. Bioorg MedChem Lett 13: 623–627.
MIRANDA MM, GONÇALVES JL, ROMANOS MT, SILVA
FP, PINTO L, SILVA MH, EJZEMBERG R, GRANJA LFAND WIGG MD. 2002. Anti-herpes simplex virus effectof a seed extract from the tropical plant Licania tomentosa(Benth.) Fritsch (Chrysobalanaceae). Phytomed 9: 641–645.
MONTEIRO JM, ALBUQUERQUE UP, LINS-NETO EMF,ARAÚJO EL AND AMORIM ELC. 2006. Use patternsand knowledge of medicinal species among two ruralcommunities in Brazil’s semi-arid northeastern region. JEthnopharmacol 105: 173–186.
MOSMANN T. 1983. Rapid colorimetric assay for cellulargrowth and survival: application to proliferation andcytotoxicity assays. J Immunol Methods 16: 55–63.
NICOLETTI I, MIGLIORATI G, PAGLIACCI MC, GRIGNANI
F AND RICCARDI C. 1991. A rapid and simple methodfor measuring thymocyte apoptosis by propidium iodidestaining and flow cytometry. J Immunol Methods 139:271–279.
OLAJIDE OA, ECHIANU CA, ADEDAPO AD AND MAKIN-DE JM. 2004. Anti-inflammatory studies on Adenantherapavonina seed extract. Inflammopharmacol 12: 196–202.
RODRIGUES LV, FERREIRA FV, REGADAS FS, MATOS DAND VIANA GS. 2002. Morphologic and morphometricanalyses of acetic acid-induced colitis in rats after treat-ment with enemas from Myracrodruon urundeuva Fr. All.(Aroeira do Sertão). Phytother Res 16: 267–272.
DMAF, BIEBER LW AND PAIVA PMG. 2009. Larvici-dal activity of lectins from Myracrodruon urundeuva onAedes aegypti. Comp. Biochem Physiol 149: 300–306.
SAMPAIO CA, MOTTA G, SAMPAIO MU, OLIVA ML,ARAÚJO MS, STELLA RC, TANAKA AS AND BATISTA
IF. 1992. Action of plant proteinase inhibitors on en-zymes of the kallikrein kinin system. Agents ActionsSuppl 36: 191–199.
SAMPAIO EVSB. 2002. Vegetação & Flora da Caatinga.Associação Plantas do Nordeste/Centro Nordestino deInformação sobre Plantas. Recife, p. 49–90.
SANTOS JP, ARAÚJO EL AND ALBUQUERQUE UP. 2008.Richness and distribution of useful woody plants in thesemi-arid region of northeastern Brazil. J Arid Environ72: 652–663.
SHAHAT AA, EL-BAROUTY G, HASSAN RA, HAMMOUDA
FM, ABDEL-RAHMAN FH AND SALEH MA. 2008.Chemical composition and antimicrobial activities ofthe essential oil from the seeds of Enterolobium contor-tisiliquum (leguminosae). J Environ Sci Health B 43:519–525.
SOUSA MP, MATOS FJA, MATOS MEO, MACHADO MILAND CRAVEIRO AA. 2004. Constituintes químicos ati-vos e propriedades biológicas de plantas medicinais bra-sileiras. Edições UFC. Fortaleza, 448 p.
SOUZA SM, AQUINO LC, MILACH ACJR, BANDEIRA MA,NOBRE ME AND VIANA GS. 2007. Anti-inflammatoryand antiulcer properties of tannins from Myracrodruonurundeuva Allemão (Anacardiaceae) in rodents. Phyto-ther Res 21: 220–225.
STRASSER A, O’CONNOR L AND DIXIT VM. 2000. Apop-tosis signaling. Annu Rev Biochem 69: 217–245.
SUFFNESS M AND PEZZUTO JM. 1990. Assays related tocancer drug discovery. In: HOSTETTMANN K (Ed),Methods in plant biochemistry: assays for bioactivity.Academic Press, London, p. 71–133.
SURH YJ, CHUN KS, CHA HH, HAN SS, KEUM YS,PARK KK AND LEE SS. 2001. Molecular mechanismsunderlying chemopreventive activities of anti-inflam-matory phytochemicals: down-regulation of COX-2 andiNOS through suppression of NF-κB activation. MutationRes 480–481: 243–268.
TORRES MR, SOUSA APA, FILHO EATS, PESSOA C,MORAES MEA, MORAES MO AND COSTA-LOTUFO
LV. 2005. Biological activity of aqueous and organicextracts of seaweeds form Ceará, Brasil. Arq Cien Mar38: 55–63.
TSUZUKI JK, SVIDZINSKI TI, SHINOBU CS, SILVA LF,RODRIGUES-FILHO E, CORTEZ DA AND FERREIRA
IC. 2007. Antifungal activity of the extracts and sa-ponins from Sapindus saponaria L. An Acad Bras Cienc79: 577–583.
An Acad Bras Cienc (2011) 83 (3)
“main” — 2011/7/13 — 11:43 — page 1058 — #14
1058 PAULO MICHEL P. FERREIRA et al.
VIANA GSB, BANDEIRA MAM AND MATOS FJA. 2003.Analgesic and antiinflammatory effects of chalcones iso-lated from Myracrodruon urundeuva Allemão. Phytomed10: 189–195.
XAVIER-FILHO J. 1993. Sementes e suas defesas contra inse-tos. Projeto multinacional de biotecnologia e alimentos.Organização dos Estados Americanos, p. 1–31.
WANG IK, LIN-SHIAU SY AND LIN JK. 1999. Inductionof apoptosis by apigenin and related flavonoids throughcytocrome c release and activation of caspase-9 andcaspase-3 in leukaemia HL-60 cells. Eur J Cancer 35:1517–1525.
